We propose the novel hypothesis that peroxides induce intestinal secretion and therefore may be a potential mediator of diarrhea in inflammatory bowel disease (IBD). Phagocytes are abundant in the intestinal mucosa in active IBD; reactive oxygen metabolites are released in large quantities by activated phagocytes in the vicinity of the intestinal epithelium. Reactive oxygen metabolites nave been postulated to mediate mucosal breakdown, but heretofore have not been implicated for mediating intestinal secretion. In preliminary studies using the T84 human colon epithelial cell line, we found that hydrogen peroxide and an organic hydroperoxide stimulated active ion transport. We also found evidence that cGMP may mediate the response through a thioldependent (possibly glutathione-mediated) mechanism. We will investigate the mechanism of peroxide-induced active ion transport in T84 monolayers in Using chambers. Active ion transport will be followed continuously by recording snort circuit current, and epitnelial integrity will be followed by measuring transepithelial resistance. We will characterize the specific ions being transported by using selective ion channel and transport blockers, ion substitution, and unidirectional tracer ion flux techniques. We will confirm our preliminary data that guanylate cyclase is activated by oxidants in T84 monolayers by measuring cGMP and cAMP formation by RIA. In whole cells we will investigate a free-radical mediated mechanism of peroxide-induced secretion by applying hydroxyl radical inhibitors, measuring lipid peroxide production, and correlating with effects on Isc and R. We will attempt to confirm or dissociate a specific role for oxidized glutathione in T84 secretion and guanylate cyclase activation. We will apply a specific glutathione oxidizing agent, diamide, and compare its effects with those of peroxides on GSSG formation, cGMP production, and Isc responses in T84 monolayers. Soluble and particulate guanylate cyclase will be prepared in a cell-free system to investigate directly oxidant-, thiol-, or glutathione- mediated activation of guanylate cyclase as suggested by whole cell studies. Finally, effects peroxides on breakdown of paracellular resistance, cell lysis, and morphologic integrity of the monolayer will be examined by unidirectional flux of labeled mannitol and inulin, release of cytosolic tracers, and scanning electron microscopy. Our ultimate goal will be to define specific interactions of reactive oxygen metabolites with intestinal epithelial cells and their pathophysiologic consequences.